Storage-stable hemostatic transfusion suspensions of blood platelets, glucose, magnesium chloride and certain prostaglandins

ABSTRACT

The invention provides storage-stable aqueous isotonic saline suspensions containing mammalian blood platelets glucose, magnesium chloride, and certain prostaglandins. The hemostatic function of the platelets is preserved in these suspensions.

United States Patent Sekhar, N. C. J. Medicinal Chem. 13: 39- 44 (1970)Eflnventor Neelkant C. Selghar Kalamazoo, Mich. App]. No. 10,318 FiledFeb. 10, 1970 Patented Dec. 21, 1971 Assignee The Upjohn CompanyKalamazoo, Mich.

STORAGE-STABLE HEMOSTATIC TRANSFUSION SUSPENSIONS OF BLOOD PLATELETS,GLUCOSE, MAGNESIUM CHLORIDE AND CERTAIN PROSTAGLANDINS l Claim, NoDrawings US. Cl 195/].8,

feet of Eight Prostaglandins on Platelet Aggregation."

Elkeles, R. S. et al., Lancet 2: l 1 l (1969) Prostaglandin E1 and HumanPlatelets."

O Brien, .1. R. Lancet 1: 149 (1968) Prostaglandins and Platelets."

Crevelds, von et al., Nature 218: 361- 362 (I968) lnfluence of theProstaglandins E1 and E2 on Aggregation of Blood Platelets.

Chandrasekhar, N. Blood 30: 554 (1967) Inhibition of PlateletAggregation by Prostaglandins."

Emmons, P. R. et al., BR. Med. J. 2: 468-472 (1967) Effect ofProstaglandin E1 on Platelet Behavior in vitro and in vivo" PrimaryExaminer-Shep K. Rose Attorneys-Edward G. Jones and John Kekich IABSTRACT: The invention provides storage-stable aqueous STORAGE-STABLEHEMOSTATIC TRANSFUSION SUSPENSIONS OF BLOOD PLATELETS, GLUCOSE,MAGNESIUM CHLORIDE AND CERTAIN PROSTAGLANDINS BACKGROUND OF THEINVENTION A crude mixture, called prostaglandin, was reported by vonEuler, Arch. Exp. Path, Pharm Abs. 175-78 (1934); 181 (1936); J. Physiol72,74 (1931); 81,102 (1934); 84,21 (1935); 88,213 (1936); and Klin,Wschr 14, 1182 (1935). More recently essentially pure crystalline PGF(PGF or PGF a) has been isolated, British Pat. No. 851,827 and ActaChemica Scandinavica 14, 1693 (1960). Microbiological conversions ofunsaturated fatty acids with mammalian glandular tissue are described ain U.S. Pat. Nos. 3,290,226 and 3,296,091. In the latter patent PGF (PGFor PGF,a) is designated as 7-[3a, 5a-dihydroxy-2-(3-hydroxy-1-ocetenyl)-cyclopentyl1-heptanoic acid.

The PGF-type prostaglandins are characterized by the presence of thehydroxyl group at the five-position in the cyclopentane ring. Thedesignation PGF a shows the configuration of the hydroxyl at thefive-position. Various other members of the PGF-type are known and arenamed either systematically or in terms of their relationship to PGF.Illustrative thereof are PGF a or7-[3a-5a-dihydroxy-2-(3-hydroxy-1-octenyl)-cyclopentyll-5-heptenoicacid, PGF or 7-[3a,5adihydroxy-2-( 3-hydroxy-l ,5-octadienyl)-cyclopentyl]-5-heptenoic acid, and dihydro PGF,a or 7-[3a,5a-dihydroxy-2-(3- hydroxyoctyl)-cyclopentyll heptanoic acid. Details ofpreparations from available materials are disclosed for dihydro PGF a,PGF a, and PGF a in Biochimica and Biophysica Acta, 84, 707 (1964), andfor PGF a in U.S. Pat. No. 3,069,322. Bergstrom, Carlson and Weeks,Pharmacological Reviews, Vol. 20, No. 1, P. 1 (1968) review TheProstaglandins.

In U.S. Pat. No. 3,290,226 PGE compounds are described including PGE,,PGE and PGE The PGE Series is characterized by the presence of the ketogroup at the five-position in the cyclopentane ring. More recently,Ramwell et al., Prostaglandins" in Progress in the Chemistry of Fats andother Lipids, Vol. 9 edited by R. Holman, pp. 73-115, Pergamon Press,Oxford, 1967 refer to prostaglandin PGE as I la, l5(S)-dihydroxy-9-oxo-l3-trans-prostenoic acid, PGE as 1 la, l5( S )-dihydroxy-9-oxo-5-cis, l3-trans-prostadienoic acid and PGE as 1111,15(S)-dihydroxy-9-oxo-5-cis,13-trans, 17- cis-prostatrienoic acid.

PGE l5-formate is prepared as follows: A solution of sodium carbonate(50 mg.) in 7.5 ml. of dry formic acid is added to 250 mg. of PGE,. Thismixture is stirred under nitrogen at 25 C. for 2 hours. The reactionmixture is evaporated under reduced pressure. Benzene is added to theresidue and the mixture is again evaporated under reduced pressure. Theresidue is then chromatographed on 50 g. of silica gel (acid washed topH 4; 100-200 U.S. mesh; Mallinckrodt Silicar CC-4), eluting with 2.5 1.ofa gradient of 25 to 75 percent ethyl acetate-isomeric hexane mixture(Skellysolve B), and collecting l00-m1. fractions. Eluate fractions 9,10, and 11 are combined and evaporated to dryness under reduced pressureto give 99 mg. of PGE 15-formate. 8-iso-PGE, is described by Daniels, etal., J. Am. Chem. Soc. 90, 5894 (I968); dl-8-Iso- PGE,, methylester isdescribed by Schneider et al., .I. Am. Chem. Soc. 90, 5896 (1968); andll-dehydro-PGE a is described by Lands et al., J. Biol. Chem. 243, 4104(1968).

Excepting PGE these prostaglandins are to be construed as includingoptically active compounds of the natural configuration and racemiccompounds. These compounds are known in the art or are available bymethods known in the art. See U.S. Pat. No. 3,296,091; Rec. Trav. Chim.85; 1233 (1966), Ibid. 87, 461 (1968); .I. Am. Chem. Soc. 90, 5895(1968). For the racemic compounds, see, for example, ChemicalCommunications, pp. 303-305 (1969).

Pharmaceutically acceptable salts for example, those of a1- kali metalsand alkaline earth bases, such as the sodium, potassium, calcium andmagnesium salts; those of ammonia or a basic amine such as mono-, di-,and triethyl amines, benzylamine, heterocyclic amines such as piperidineand morpholine, and amines containing water-solubilizing or hydrophilicgroups such as triethanolamine and phenylmonoethanolamine are disclosedin U.S. Pat. No. 3,296,091. Carboxylate esters such as methyl, ethyl,cyclohexyl and the like having no more than eight carbon atoms areformed by the usual methods, e.g., reaction with diazomethane or similardiazohydrocarbons as in U.S. Pat. No. 3,296,091.

Biological studies of the prostaglandins, for example, actions on smoothmuscle, reproductive system, nervous system, cardiovascular system, andrelationship to lipid and carbohydrate metabolism, and miscellaneouseffects are summarized by Bergstrom et al.; The Prostaglandins: A Familyof Biologically Active Lipids, Pharmacological Reviews, Vol. 20, No. l,P.1 et sequitur, 1968, The Williams and Wilkins Company.

Blood platelets when stored for a few hours, and then infused, lose mostof their ability to perform the hemostatic function. Consequently, allthrombocytopenias (congenital as well as drug-induced) must be treatedwith freshly prepared platelet suspensions. The instability of thephysiological properties of platelets results in total waste of unusedplatelet suspensions and also presents a major problem in the treatmentof thrombocytopenias. Inhibition of platelet aggregation by someprostaglandins, including POE has been demonstrated in variousinvestigations. Kloeze, .l., Nobel Symposium 2, Prostaglandins, Ed. 5.Bergstrom and B. Samuelson, pp. 24l252. Almquist and Wiksell, Stockholm(1967). Emmons, P. R., Hampton, J. R., Harrison, M. .l. R., Honour, A.J., and Mitchell, J. R. A., Brit. Med. J., 2, 468 (I967). Chandrasekhar,N., Blood, 30, 554 (1967). However, until the present invention,storage-stable suspensions of platelets both human and animal were notavailable.

SUMMARY OF THE INVENTION This invention relates to the preservation ofthe hemostatic function of blood platelets. More specifically, itrelates to storage-stable aqueous suspensions of mammalian bloodplatelets, for example, human and animal such as rabbit, rat, andmonkey. The aqueous suspensions consist essentially of said plateletsand a prostaglandin selected from the group consisting of PGE.; POE15-formate; 8-Iso-PGE,; dI-8-lso-PGE methylester; and l l-dehydro-PGF,a.

The aqueous suspensions of the platelets are preferably in the form ofplatelet-rich plasma or an aqueous suspension in 0.9 percent NaCl. Theamount of prostaglandin for preservation of the hemostatic function ofthe platelets is within the nontoxic effective range from about 0.025ug./ml. to about 1.0 mg./ml.

Additional ingredients complementing the preservative action of theprostaglandin component are glucose from about I to about 2 mg./ml. andfrom about 2.5 to about 10 milliequivalents of Mg in the form ofmagnesium chloride. In the suspensions the hemostatic activity of theplatelets is preserved permitting their use in clinical conditions suchas idiopathic thrombocytopenia and drug-related thrombocytopeniasometimes accompanying therapy with, for example, chloramphenicol,mustard-type cytotoxic agents, and cobalt treatments. Normally the humanplatelet count is from about 300,000 to about 500,000 per ml. In severethrombocytopenia it may be depressed to about 50,000 per liter or lowernecessitating infusion of 5 to 6 liters of preserved platelet suspensioncontaining the effective nontoxic amount of prostaglandin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS A model system involving adecrease in the number of platelets in rats is used to show the storagestability of blood platelets in combination with the prostaglandincomponent.

Platelet counts of Platelet-Rich Plasma obtained from Rats X- Irradiatedat four different levels (three animals per group; Mean Counts expressedas Xl /mm.

No. of days following X- lrradiation X-lrradiation Level 100 R 200 R 300R 400 R Day 5 1,964t80 1,487t33 1,151t162 1,2422161 Day 7 1,8091-731,372t69 1109-552 9851140 Day 9 1,1271-137 880x87 593:97 471x105 Day II1,8161232 1,3Z1t91 1,17421269 927195 Three groups (12 rats/group) ofmature male Sprague- Dawley rats are X-irradiated at 300 R. On the 6thday following X-irradiation, fresh platelet-rich plasma (PRP) iscollected by centrifuging citrated blood (from another set ofnonirradiated Sprague-Dawley rats) at 1200 r.p.m. for 10 minutes. Onealiquot of the PRP is incubated with PGE (I00 ug./ml.) for 48 hr. at 5.Another aliquot of PRP is incubated with an equal volume ofphysiological saline (positive control). On the 8th day followingX-irradiation, another sample of fresh PRP is obtained. One group of 12rats (negative controls) is given tail vein injections of the freshlyprepared PRP (four injections of 2 ml. each, equally spaced within 8hr.). Another group (positive controls) is given similar injections ofsaline-incubated PRP and the third group (experimental) is given PGE-incubated PRP. All rats are sacrificed 24 hr. after infusion and theplatelet aggregation (collagen-induced) of their PRP samples isdetermined using the Aggregometer. Results show that negative controlswith fresh platelets give the maximum aggregation values and thepositive control group with saline-incubated PRP shows the poorestaggregation values compared to the positive control group, indicatingthat PGE preserves the hemostatic function of stored platelets.

Platelet-Aggregation in Thromocytopenic Rats after PRP- InfusionTreatment Collagemlnduced Aggregation in 5 Min.

Negative Control 8. 1:0.7

Positive Control 3.0105

Experimental (PGE incubated PRP) 3.710.!

INHIBITION OF PLATELET AGGREGATION to yield a 10 mg./ml. solution.Acidic solutions are then diluted to l mg./ml. concentration, with a 0.2mg./ml. sodium carbonate solution; esters are diluted to a concentrationof l mg./ml. with 0.9 percent sodium chloride. Subsequent dilutions ofall prostaglandins are accomplished with 0.9 percent sodium chloride;freshly prepared solutions only are used in the experiments.Platelet-rich plasma (PRP) is used as the source oflplatelets. Freshldrawn cit'rated blood samples from adult mae Sprague-Daw ey rats(Spartan strain) are centrifuged at 1200 r.p.m. for 10 min. to obtainrat PRP. Three aggregation inducers are used: (I) 0.25M calcium chloridesolution; (2) 50 mg./ml. adenosine diphosphate solution (ADP) freshlyprepared using Ringer's solution; (3) collagen suspension made up inTyrode solution without calcium.

Two methods are employed to measure platelet aggregation parameters: amodification of Chandler's visual technique using a revolving plasticloop and a photo-optical method using an Aggregometer (ChronologCorporation, Broomall, Pa.). For the Chandler's loop method, 0.8 ml.volume of PRP is taken up in a plastic loop (Transflex No. 8, 3MCompany), 0.1 ml. of a 1 mg. per ml. solution of the prostaglandin isadded to the plasma and the loop is rotated at 12 r.p.m. for 1 minute toassure proper mixing. An 0.] ml. volume of 0.25M calcium chloride isthen injected onto the plasma layer at one end of the loop and threestop watches and the loop are started simultaneously. Three end pointswhich occur in succession are recorded: l aggregation of plateletsvisible to the naked eye (VA); (2) appearance of snowstormlike plateletclumps (SS); and (3) a well-packed platelet disc or head at one end ofthe plasma surface (PH). Similar experiments are carried out usingphysiological saline in place of prostaglandin solution, to obtaincontrol values. If VA obtained with prostaglandin is greater than 10seconds from that of the controls, successive dilutions ofprostaglandins are made and tested. The lowest concentration ofprostaglandin that prolongs VA for more than 10 seconds is tested onthree separate rat PRP samples and the mean and standard error of VA, SSand PH are recorded.

Platelet aggregation phenomenon induced by ADP and collagen is studiedin the Aggregometer. Freshly obtained rat PRP (0.8 ml.) is mixed with0.1 ml. of the prostaglandin solution in an Aggregometer cuvette for 1minute. O.l ml. of the collagen suspension is pipeted into this cuvetteto induce aggregation. Platelet aggregation/adhesion that takes place inthe cuvette produces changes in optical density which are recorded on aBausch and Lomb VOM 6 recorder. The height of the curve recorded istaken as the aggregation index. Identical experiments are done using PRPfrom control animals also. If the aggregation index for the experimentalsamples is less than percent of that of the controls, prostaglandin (atthat particular concentration) is considered active.

All prostaglandins are initially tested in the Chandler's loop todetermine the lowest concentration that is active. The compound is againtested in the Aggregometer at the lowest active concentration, againstADP and collagen.

lclaim:

1. A storage-stable aqueous suspension in isotonic saline with effectivecomplementary hemostatic activity preserving concentrations of glucoseand magnesium chloride for transfusion consisting essentially ofmammalian blood platelets and a prostaglandin selected from the groupconsisting of P615 PGE,-l5 formate; 8-Iso-PGE,, dl-8-Iso-PGEmethylester', and l l-dehydro-PGF,a, the amount of the prostaglandinbeing within the nontoxic effective range from about 0.025 ug./ml. toabout 1 mg./ml. of the suspension.

